Composition of matter.



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GO'MPOSITEDN F llUJlJTEZEt.

Man-Mm stated, viz

Nutrient broth, 1000 cubic centimeters;

Agaragar, 30 grains;

Litmus solution, from 120 to 150 cubic centimeters;

Malachite green, 1? cubic centimeters of a one per centsolution;

Crystal violet, aqueous solution of, 10 cubic centimeters of a one-tenthper cent. solution.

The above represen s the best relative proportions of the particularsamples of the ingredients 1 am using at present; other samples,particularly of crystal violet and malachite green, must have theirpropon tions determined according to then: combining power andadaptability, which vary widely with the difierent commercial grades ofthese materials. The range of proportions of the various ingredients forone liter of broth, for example, has been found to be: agar-agar, 10 to50 grams; litmus solution, 50 to 300 cc.; crystal violet, to 4E0 cc. ofa one-tenth per cent. solution; malachite green, to 80 cc; of acne percent. solution.

The nutrient broth. is to be, preferably, sugar-freebeet broth alkalineto litmus'to the extent of about 4 cubic centimeters normal sodiumcarbonate beyond the neutral point. It beet broth in 'sutlicientquantity is still deficient-in nutrition, an additional nutrient, suchas nut-rose or 'glycerin-either or both.to the amount of 5 to 10 gramsof the former or 50. cc. of the latter, may be employed or added.

The agar-agar should be finely divided, melted, Well filtered, andclarified.

lhe litmus solution may be substitutedby any kind of indicator insuliicient quantity to show reaction produced by Specification ofLetters catent.

Patented Nov. it'll, rem.

Application filed Qctober 5,1912. Serial No. $585,012.

germs. Lactose or milk sugar, to the amount of (3 to 8 grams, may withthe indicator.

The crystal violet and the malachite green (the latter is also oftentermed benzaldehyde green) may be used in any suitable proportion orrelation either before or after these ingredients, or their resultantproducts, enter the medium. These ingredients are derivatives oftriphenylemethane and are both basic colors. Grystal violet is. atriamino derivative of triphenylmetliane, and malachite green a diaminoderivative of triphenylmethane.

Tubing, putting about 8 cubic centimeters in each sterile test tube. Thesterllization may be fractional or discontlnued.

be used in connection Gil In compounding the medium I mix the solutionsof crystal violet and malachite green in suitable proportions, allowtime for reaction, add them directly to the medium and the Wholethoroughly. in the majority of cases the best proportion is 1 cc. ofcrystal violet With 16' to 20.

cc. (usually 1? cc.) of malachite green; that is to say, 10 cc. of aone-tenth per cent. solution of crystal violet and 16 to 20 cc. of a oneper cent. solution of malachite green, or the equivalent'of these."These solutions are aqueous and should be freshly and accurately madefor each medium, as they tend to deteriorate. ll therefore take 10 cc.of a one-tenth per cent, aqueous solution of crystal violetand. mix itwith 16 to 20 cc. (preferably 17 cc.) of a one per cent. solution ofmalachite green and allow it to stand for one or two hours and heat itjust a little in the Water bath before adding it to the medium, mixthoroughly and tube the medium at once, putting about 8 co. in the testtube. ll sterilize by fractional or discontinued sterilization forminutes immediately after lllll 'WZltQIY and spread It is best .to learnthe'characteristics of the typhoid colony as they are manifested in thismedium by studying known typhoid or para-- typhoid colonies undervarious circum-v stances as they appear in the medium from day to day.It is also best to use tWo or three Petri dishes so as to check results.I always aim to get not more than one hundred colonies in each dish. Inpreparing the medium for inoculation, I place the tubes 01' themediumina ring stand in the-Water bath with suflicient ater to cover themedium and boil until the latter is entirely melted, but hotlonger-threeminutes is about the limit. I then cool down the Water bath to aboutdegrees C, and allow it to cool gradually to about $7 degrees C. androll. the dish wellso as to distribute the germs evenly. (Note careshould be'taken not to pour any sediment' from the bottom of the testtube into the Petri dish, as sediment interferes greatly with themicroscopic work.) .The medium of the present invention maybe inoculatedthe same as any other agar-agar media.

After the medium described has been cooled. down to about d7 degreescentigrade, it is ready for inoculation. Inoculation, is made according.to the material to be tested, its condition, and the results aimed at.In general, inoculation of this medium is made as in any-other agar-agarmediunn The medium of the present invention is'designed to'be' usedchiefly fortesting ater, to find out as nearly as possible the number oftyphoid orparatyphoid germs present in the Water. One of the Ways ofusingthis medium is to put the desired amount of suspected Water into asterile Petri 'dish by means of a graduated sterile pipetteinsertedunder the raised cover of the dish, and then the mouth of the tubecontaining cooled me dium is similarly inserted and the medium pouredinto and quickly'mixed With the by tilting the dishand rolling-itgently. The dish is then placed on a level surface'until the medium issolidified? and the germs contained therein become incubatedL Tofacilitate the counting of the germs contained in the \vater'thesiispected water may be poured into the tube containing the cooledmedium. The tube is to be held vertically in the hand and whirled untilthe water is thoroughly and evenly mixed in the medium. -'lhe mixtureisthen poured out and distributed as described. This methodgives an evendistribution-of the germs and thus facilitates their counting andisolating. I The inoculation of fluids other than Water is n'iade.preciscly the same way after proper dilutions have been made. Amicroscopic exan'. .tion of a drop of the suspected fluid number.

inhibitive ingredients,

imes g'ives a fair idea of the amount of dilution necessary. Withtherexact amount of dilution known the number of germs present in thedrig'inal fluid is readily ascertained. A series of dilutions may bemade up to any These dilutions are to be made by means of sterile dishesand sterile graduated pipettes, ascending from the lowen dilutions tothe higher. In this Way milk, blood, sewage, and other fluids may betested.

The typhoid and paratyphoid colonies in this medium are peculiar tothemselves and dilferent'fiom all others. ,The medium of the presentinvention tendsto sift out germs from all others growing in the fluidwhich is being tested. Plain media, tin "or. agar-agar and those willshow the Whole number-of germs in the fluid tested;-While a, medium likethe Drigalski-Conradi will showthe number of colon and other acidforminggerms present, .because they ferment the milk sugar in that medium, and

the. colonies appear red, for the litmus of the medium shows the resenceof the-acid they form. Typhoidand are chiefly alkaline inreaction andferment milk ,sugar'but 'feebly, and then only after a considerableperiod had elapsed' These colonies do not therefore turn red like thecolon on the Drigalski-Conradi medium and are not characteristic enoughto enable the observer to'distinguish them readily from other smallcolonies growing in the. same medium. The presentmedium increases thesize of the typhoid and paratyphoid coloby proper nutrients inthemediuzn, Whiehhelps m'ateriall'y to distinguish them from others; thetyphoid and paratyphoid colonies take on a "peculiar characteristiccolor scheme, forming concentric rings or hands of different colors.

While media containing several of the ingredients herein described havebeen used before, nomedium has, to my knowledge, ever been-produced intoand. malachite green entered either before or after introduction into.to be used-as an aid in the detection, isolation, or identification ofthe germs of ty phoid or of paratyphoid fevers in fluids or solids.

such as gelanot contai-mng' paratyphoid germs which crystal violetamedium adapted serving the right to make such alterations and changes inthe compounding of the same as fairly fall Within thespirit and myinvention, I claim:

1. A composition'of matter for use as an aid in the detection, isolationor identificascope of tion of the germs of typhoid or paratyphoid fevercomprising crystai violet 5 to tlicubi'c centlmeters and malachite green10 to 80 cubic centimeters-to a predetermined quantity of agar-agarmedium, as one liter.

2. A'co'mposition oit matter comprising a mes-noes crystal violet andmalachite in suitable proportions, in coni- Z1 10 to 50 grams ofagar-agar esleteiiniiiedquantity of nutrient tel,

' tion of matter consisting, as i 'Cl, of a suitable quantity of f jroiuct of crystal violet and 1, mixed. in thBYPIOPGL proeombiniug power,and added or medium comprising about ltl'lfllli? broth and 50 to 300"mus solution, or equivalent c hi cicnt quantity to show wel by germs.

oositioii' of matter comprising *1 about one liter, agar-agar unis, anindicator in sufficient show reaction produced by ween germs, and asolutionoi. crystal violet and malachite green mixed in suitableproportions, as set forth and described.

5. The herein described composition of matter consisting of nutrientbroth 1000 cubic centimeters, agar-agar 30 grams, litmus solution fromto 150 cubic centimeters, crystal violet 10 cubic centimeters oi aone-tenth per cent. solution, and malechite green 1'? cubic centimetersof a one per cent. solution, substantially as described.

In testimony whereof I have signed my name to this specification in thepresence of two subscribing witnesses this 30th day of September, 191st.

Alt-THUR lVlLLl'Al /l FISHER.

Wituesse HARRY FISHER, HoWARD E. COLBY.

